The present invention relates to a method for the taxonomic identification of pathogenic microorganisms and the detection of their proteinaceous toxins.
Pathogenic microorganisms, particularly pathogenic bacteria which either occur naturally or which have acquired virulence factors, are responsible for many diseases which plague mankind. Many of these bacteria have been proposed as biowarfare agents. In addition, there is also the risk and likelihood that nonpathogenic microbes could also be used as pathogens after genetic manipulation (e.g., Escherichia coli harboring the cholera toxin).
Typical pathogenic bacteria include those responsible for botulism, bubonic plague, cholera, diphtheria, dysentery, leprosy, meningitis, scarlet fever, syphilis and tuberculosis, to mention a few. During the last several decades, the public perception has been one of near indifference in industrialized nations, principally because of successes that have been achieved in combating these diseases using antibiotic therapy. However, bacteria are becoming alarmingly resistant to antibiotics. In addition, there have been recent revelations of new roles that bacteria perform in human diseases such as Helicobacter pylori as the causative agent of peptic ulcers, Burkholderia cepacia as a new pulmonary pathogen and Chlamydia pneumoniae as a possible trigger of coronary heart disease. Apart from those pathogens, various socioeconomic changes are similarly contributing to the worldwide rise in food-borne infections by bacteria such as Escherichia coli, Salmonella spp., Vibrio spp., and Campylobacter jejuni. 
Potential infections are also important considerations in battlefield medicine. A number of bacterial pathogens, including Bacillis anthracis and Yersinia pestis and their exotoxins, have been used as weapons. And there is always the risk that nonpathogenic microbes can be engineered to be pathogenic and employed as biowarfare agents.
Pathogenic microorganisms are also of concern to the livestock and poultry industries as well as in wildlife management. For example, Brucella abortus causes the spontaneous abortion of calves in cattle. Water supplies contaminated with exotoxin-producing microorganisms have been implicated in the deaths of bird, fish and mammal populations. More recently, mad cow disease has been traced to the oral transmission of a proteinaceous particle not retained by filters. Thus, there is clearly a need for rapid and inexpensive techniques to conduct field assays for toxic proteins and pathogenic microorganisms that plague animals as well as humans.
As a general proposition, bacterial contamination can be detected by ordinary light microscopy. This technique, however, is only of limited taxonomic value. The investigation and quantitation of areas greater than microns in size are difficult and time consuming. Many commercially available systems rely on the growth of cultures of bacteria to obtain sufficiently large samples (outgrowth) for the subsequent application of differential metabolic tests for species (genus) identification. However, techniques requiring bacterial outgrowth may fail to detect viable but nonculturable cells. To the contrary, the growth media employed may favor the growth of bacteria with specific phenotypes.
More sensitive and more rapid typing schemes are described in xe2x80x9cStrategies to Accelerate the Applicability of Gene Amplification Protocols for Pathogen Detection in Meat and Meat Productsxe2x80x9d by S. Pillai and S. C. Ricke (Crit. Rev. Microbiol. 21(4), 239-261 (1995)) and xe2x80x9cMolecular Approaches for Environmental Monitoring of Microorganismsxe2x80x9d by R. M. Atlas, G. Sayler, R. S. Burlage and A. K. Bej (Biotechniques 12(5), 706-717 (1992)). Those techniques employ the polymerase chain reaction (PCR) for amplification of bacterial DNA or RNA, followed by nucleic acid sequencing to detect the presence of a particular bacterial species. Such general amplification and sequencing techniques require technical expertise and are not easily adaptable outside of specialized laboratory conditions. PCR-based techniques utilize the inference of microbial presence since these techniques provide only a positive analysis whenever an intact target nucleic acid sequence, not necessarily a microbe, is detected. PCR is also unable to detect the presence of toxic microbial proteins. Moreover, the detection of specific microorganisms in environmental samples is made difficult by the presence of materials that interfere with the effectual amplification of target DNA in xe2x80x98dirtyxe2x80x99 samples.
Mass spectral analysis of volatile cell components (e.g., fatty acids) after sample lysis or pyrolysis has been used for the detection of bacteria and viruses. One description of the methods used to detect microorganisms with this method can be found in xe2x80x9cCharacterization of Microorganisms and Biomarker Development from Global ESI-MS/MS Analyses of Cell Lysatesxe2x80x9d by F. Xiang, G. A. Anderson, T. D. Veenstra, M. S. Lipton and R. D. Smith (Anal. Chem. 72 (11), 2475-2481 (2000)). Unfortunately, identification of the analyte is unreliable as the compositions of a microbe""s volatile components change depending upon different environmental growth conditions.
Another approach utilizes immunochemical capture as described in xe2x80x9cThe Use of Immunological Methods to Detect and Identify Bacteria in the Environmentxe2x80x9d by M. Schlotter, B. Assmus and A. Hartmann (Biotech. Adv. 13, 75-80 (1995)), followed by optical detection of the captured cells. The most popular immunoassay method, enzyme-linked immunosorbent assay (ELISA), has a detection limit of several hundred cells. This is well below the ID50 of extremely infectious bacteria such as Shigella flexneri. Piezoelectric detection techniques, such as those described by xe2x80x9cDevelopment of a Piezoelectric Immunosensor for the Detection of Salmonella typhimuriumxe2x80x9d by E. Prusak-Sochaczewski and J. H. T. Luong (Enzyme Microb. Technol. 12: 173-177 (1990)) are even less sensitive having a detection limitation of about 5xc3x97105 cells. A recent report entitled xe2x80x9cBiosensor Based on Force Microscope Technologyxe2x80x9d by D. R. Baselt, G. U. Lee and R. J. Colton (Biosens. and Bioelectron. 13, 731-739 (1998)) describes the use of an atomic force microscope (AFM) to detect immunocaptured cells; this method has little utility outside a laboratory setting and when the sample volumes are large. Immunoassays are also presently used in the trace analysis of peptides and proteins.
Moreover, the prior art has made extensive use of immobilized antibodies in peptide/protein/microorganism capture. Those techniques likewise involve significant problems because the antibodies employed are very sensitive to variations in pH, ionic strength and temperature. Antibodies are susceptible to degradation by a host of proteolytic enzymes in xe2x80x9cdirtyxe2x80x9d samples. In addition, the density of antibody molecules supported on surfaces (e.g., microwell plates or magnetic beads) is not as high as is frequently necessary. A good summary of the state of the art, still up-to-date, is xe2x80x9cMicrobial Detectionxe2x80x9d by N. Hobson, I. Tothill and A. Turner (Biosens. and Bioelectron. 11, 455-477 (1996)).
Medical and military considerations call for better toxin and pathogen detection technologies. Real-time assessment of battlefield contamination by a remote sensing unit is necessary to permit and facilitate rapid diagnosis for administration of appropriate counter-measures. A microbe/toxic protein sensor useful in such situation requires the ability to globally discriminate between pathogens and non-pathogens. In addition, such techniques require high sensitivity when less than 100 cells are present and analysis that can be completed in the field in less than 15 minutes. Such techniques should be able to recognize pathogens and provide some assessment of strain virulence or toxigenicity.
To date, common approaches used for the identification of pathogenic microorganisms and their proteinaceous toxins have employed immunological methodologies. Immunological methods suffer from the sensitivity of antibodies toward pH, ionic strength, and temperature; the antibodies themselves are subject to proteolysis and require careful storage conditions. To overcome these problems the present invention describes the capture of microorganisms and their proteinaceous toxins using non-antibody based ligands. It is accordingly an object of the present invention to provide a method for taxonomically evaluating microbes and proteins that overcome the foregoing disadvantages of technologies that depend upon antibodies.
It is a more specific object of the invention to provide a method for taxonomically evaluating microbes and proteins that has the capability of discriminating between specific microbial species, pathogens and nonpathogens, and can be likewise used to identify microbial proteins of diagnostic utility.
The present invention demonstrates the ability of heme compounds, siderophores, polysaccharides and peptides to bind to pathogenic microorganisms and their proteinaceous toxins; taxonomic identification of a microorganism is attained thorough analysis of the number and kind of ligands to which it binds. The development of this method was done to overcome the aforementioned limitations of antibody-based technologies. The concept of the present invention resides in a method for the taxonomic identification of microorganisms in which microbes are captured through the binding of microbial receptors to specific ligands. A microorganism-containing sample is contacted by the ligand, with the ligand being either tethered to a surface or conjugated to a marker. The target microbe (bacteria, virus, fungi, protozoa, rickettsiae, or other cell) or proteinaceous material (toxin) is then separated from the non-binding sample components and unbound ligand as by washing, magnetic separation or chromatography. Finally, the sample is interrogated by an appropriate method to determine if the ligand has been bound to the target by detecting signals endogenous to the target or marker.
Electromagnetic radiation is one method used to detect the presence of metabolites characteristic of living microbes, e.g., reduced pyridine nucleotides or other fluorescent metabolites, other biomolecules, e.g., notably tryptophan or tyrosine in proteins, or incorporated dyes for the detection of the presence of the captured microorganisms and/or toxins in accordance with the practice of the invention. For example, if the ligand contains a fluorescent dye, the sample will fluoresce after washing, since the ligand is bound to the cells and the excess is washed away. Other markers, including luminescent, phosphorescent, radioactive and/or colorometric compounds, can be conjugated to the ligand and used to identify a microbe and/or proteinaceous toxin in a similar manner.
One specific method to detect capture of microorganisms or toxic proteins is described in U.S. Pat. Nos. 5,760,406 and 5,968,766, where electromagnetic radiation is directed, for example, onto the surface of a ligand-conjugated substrate that has been treated with an analyte-containing solution as outlined above. This detection method could be used to determine if binding of an analyte has occurred. Other detection methods, appropriate for the specific kind of marker conjugated to the ligand, can also be employed to determine if the ligand has been specifically bound to a microorganism or toxic protein. An example mentioned previously uses a fluorescent dye conjugated to a ligand coupled to detection of a microbe via fluorescence characteristic of the dye after (1) contact between the microbe and ligand and (2) washing away excess dye-conjugated ligand. It is important to note that if optical methods are used to detect the captured microbe or protein the tether should not be photocleavable, e.g., the tether should be photostable.
Thus, the method of the present invention does not depend on classical antigen-antibody recognition. On the contrary, the concepts of the present invention make use of relatively inexpensive reagents in the capture of microorganisms and microbial proteins contained in the sample.
In one embodiment of the invention, sensor chips (or beads) are employed. These chips should be formed from a suitable support material such as glass or plastic substrates (e.g., poly(propylene) or poly(vinyl acetate)) that will be compatible with both the chemistries used to conjugate the linker and ligand to the surface and the detection method employed. The sensor chip is formed of a patterned array defining a plurality of sections on the surface of the sensor chip, and each section has bonded thereto a different ligand capable of molecularly recognizing a specific microbial protein or microbial receptor, and hence the microbe itself. Microbial receptors would include, for example, proteins residing in the outer membrane of the microbial cell, pilus or flagellum, which is exposed to the aqueous environment surrounding the cell. The ligand for pathogen/protein capture bonded to the surface of the sensor chip can and should be varied. In general, such ligands may be characterized as heme compounds, siderophores, polysaccharides and anti-adhesion peptides capable of capturing a wide variety of microorganisms and toxic proteins. These ligands can thus be immobilized or bonded to the surface of the sensor chip through an appropriately sized cross-linker also having the capability of reacting with the ligands, whereby the coupling agent establishes a chemical tether between the surface of the sensor chip and the ligand capable of reaction with a variety of different microorganisms and proteins. The sensor chips and arrays (1) are exposed to a solution containing microorganisms or toxic proteins, (2) the non-binding constituents of the solution are removed, (3) followed by interrogation of the ligand-tethered surfaces to detect analyte binding. Analysis of the type or pattern of ligand-tethered surfaces found to have captured the microorganism(s), or microbial proteins not contained within intact microbial cells, can be used to taxonomically identify a microorganism or its toxic protein.
Thus, the present invention can be used rapidly to identify microorganisms without the need for growing a culture of the microorganism and then microscopically examining the culture thus produced. Likewise, low levels of toxic microbial proteins can similarly be identified. It is also unnecessary to employ enzymes or antibodies in the capture of microbial metabolites as is often used in the prior art. These, and other objects, features and advantages of the present invention will become apparent upon review of the following detailed descriptions of the disclosed embodiments and the appended claims.